Poor immunogenicity of live adenovirus recombinants expressing RSV F glycoprotein that were tested in a susceptible chimpanzee. A single susceptible chimpanzee administered Ad4-F, Ad7-F, and Ad5-F sequentially, by the enteric route, failed to develop appreciable RSV specific antibodies despite efficient replication and immunogenicity of the adenovirus vector. Cold-passaged viruses derived from a wild-type parainfluenza virus type 3 evaluated in rhesus monkeys, chimpanzees and humans. Cold-passaged (cp) parainfluenza virus type 3 candidate vaccines derived from the JS wild type virus were attenuated, immunogenic and protective in rhesus monkeys. Virus at each of the cold passages tested, i.e., cp12, cpl8, and cp45, was attenuated for the upper and lower respiratory tract. The cp45 was the most restricted in the lower respiratory tract, likely due to its greater temperature sensitivity. The attenuating mutations of cpl2 and cpl8 are non-ts. cp45 possesses additional attenuating mutations and these are ts. The cp45 retained its ts phenotype following replication in rhesus monkeys. Isolates recovered from (i) chimpanzees administered cp12 virus or (ii) humans administered the cpl8 virus lost their ts phenotype. Nonetheless, the ts+ "revertant" viruses retained the attenuation phenotype when evaluated in rhesus monkeys. This indicates that the attenuation phenotype of these cp viruses is stable after replication in primates and this component of the attenuation phenotype is specified by one or more non-ts mutations. Furthermore, it appears that the rhesus monkey can be used to assess the attenuation phenotype of isolates from simian or human vaccinees. Substitution of a single avian influenza A virus gene, PB2, into a wild-type human influenza A virus yields a reassortant that is restricted in both tissue culture and monkeys. A single gene reassortant containing the avian influenza A/Mallard/NY/78 PB2 gene and deriving its other seven RNA segments from the human influenza A/LA/2/87 (H3N2) virus was overattenuated in squirrel monkeys indicating that its restriction of replication in mammalian cells in vitro is reflected in its restriction of replication in vivo. This restriction appears to be a gene constellation affect, i.e., incompatibility of polymerase proteins derived from avian and human influenza viruses.